LOCUS       sequence-217814-        4113 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  General donor vector with T2A-H2B-TagBFP coding sequence flanking by
            restriction sites for generating knock-in donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4113)
  AUTHORS   Gu B, Posfai E, Rossant J
  TITLE     Efficient generation of targeted large insertions by microinjection 
            into two-cell-stage mouse embryos.
  JOURNAL   Nat Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166.
  PUBMED    29889212
REFERENCE   2  (bases 1 to 4113)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2018 Jun 11. pii: nbt.4166. doi: 10.1038/nbt.4166."
COMMENT     Sequence Label: EasyFusion T2A-H2B-TagBFP
FEATURES             Location/Qualifiers
     source          1..4113
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(379..1170,1171..1239))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     1002..1021
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1240..1344)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      1371..1826
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1458..1477)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1668..1689
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1952..1974
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1966..1983
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1967..1983
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1993..2012
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1993..2011
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2044..2060
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2044..2060
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             complement(2071..2769)
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     CDS             complement(2788..3165)
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             complement(3172..3225)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     primer_bind     complement(3246..3262)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3247..3263)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     complement(3292..3312)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3292..3310)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3331..3347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3331..3347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3344..3366)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3355..3371
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(3379..3385,3386..3403,3404..3409))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3424..3445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3562..3579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(join(3733..4113,1..208))
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3813..3832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag
       61 aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag
      121 ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca
      181 gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga
      241 cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat
      301 cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga
      361 gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg
      421 tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga
      481 gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc
      541 agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac
      601 tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc
      661 agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc
      721 gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc
      781 catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt
      841 ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc
      901 atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg
      961 tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag
     1021 cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat
     1081 cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc
     1141 atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa
     1201 aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta
     1261 ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa
     1321 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgcgccctg
     1381 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     1441 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     1501 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     1561 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg
     1621 atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt
     1681 ccaaactgga acaacactca accctatctc ggtctattct tttgatttat aagggatttt
     1741 gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt
     1801 taacaaaata ttaacgctta caatttccat tcgccattca ggctgcgcaa ctgttgggaa
     1861 gggcgatcgg tgcgggcctc ttcgctatta cgccagctgg cgaaaggggg atgtgctgca
     1921 aggcgattaa gttgggtaac gccagggttt tcccagtcac gacgttgtaa aacgacggcc
     1981 agtgagcgcg cgtaatacga ctcactatag ggcgaattgg agctccaccg cggtggcggc
     2041 cgctctagaa ctagtggatc cttaaccgcc attaagcttg tgccccagtt tgctagggag
     2101 gtcgcagtat ctggccactg ccacctcgtg ctgctcgacg taggtctcgt tgttggcctc
     2161 cttgattctt tccagtctgt agtccacata gtagacgcca ggcatcttga ggttcttagc
     2221 gggtttcttg gatctatatg tggtcttgat gtttgcgatc agatggctcc cgcccacgag
     2281 cttcagggcc atgtcgtttc tgccttccag gccgccgtca gcggggtaca gcgtctcggt
     2341 gaaggcctcc cagccgagtg ttttcttctg catcacaggg ccgttggatg tgaagttcac
     2401 ccctctgatc ttgacgttgt agatgaggca gccgtcctgg aggctggtgt cctgggtagc
     2461 ggtcagcacg cccccgtctt cgtatgtggt gactctctcc catgtgaagc cctcagggaa
     2521 ggactgcttg aagaagtcgg ggatgccctg ggtgtggttg atgaaggtct tgctgccgta
     2581 gaggaagcta gtagccagga tgtcgaaggc gaaggggaga gggccgccct cgaccacctt
     2641 gattctcatg gtctgggtgc cctcgtaggg cttgccttcg ccctcggatg tgcacttgaa
     2701 gtgatggttg tccacggtgc cctccatgta cagcttcatg tgcatgttct ccttaatcag
     2761 ctcgctcatg gtggcgaccg gtggatcctt agcgctggtg tacttggtga tggccttagt
     2821 accctcggac acggcgtgct tggccaactc cccaggcagc agcaggcgca cggccgtctg
     2881 gatctccctg gaggtgatgg tcgagcgctt gttgtaatgc gccaggcggg aagcctcacc
     2941 tgcgatgcgc tcgaaaatgt cgttcacaaa cgaattcatg atgcccatgg ccttggacga
     3001 aatgccggtg tcagggtgga cctgcttcag aaccttgtac acatagatgg aatagctctc
     3061 cttgcggctg cgcttgcgct tcttgccgcc tttcttctgc gccttagtca ccgccttctt
     3121 ggagcccttt ttcggggcgg gagcagactt cgctggctct ggcataccgg tagggccggg
     3181 attctcctcc acgtcaccgc atgttagaag acttcctctg ccctcgaatt cgatatcaag
     3241 cttatcgata ccgtcgacct cgaggggggg cccggtaccc agcttttgtt ccctttagtg
     3301 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3361 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3421 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3481 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3541 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3601 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3661 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3721 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3781 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3841 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3901 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3961 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4021 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4081 agcagccact ggtaacagga ttagcagagc gag
//
